The present invention relates to new amphiphilic fluorine derivatives having telomeric structures, methods for their preparation, and biomedical applications.
It relates particularly to the synthesis of biocompatible fluorinated amphiphilic derivatives useful in formulations for pharmaceutical, cosmetic, veterinary or phytosanitary use, or in biology and medicine, and other uses, for example, as prodrugs, or in preparations capable of acting as gas carriers for biomedical use, as contrast agents, or media for the transport and targeting of drugs.
It is well known that cells of human tissues and organs die when they are deprived of the oxygen supplied by blood, as in hemorrhages, severe anemia, or cerebral or myocardial ischemia. Until now, the only efficient way of replacing the necessary oxygen was by blood transfusion; however, transfusion is inappropriate in some pathological or clinical cases; moreover, it almost always presents certain immunological and infectious risks. Research was therefore undertaken aiming at the preparation of satisfactory in vivo oxygen carriers which could not only replace the erythrocyte function to transport and deliver oxygen, but also be used in situations where the administration of red blood cells is ineffective or contra-indicated.
Substances capable of carrying oxygen in a dissolved form are known: they are the fluorocarbons, which are the best-known solvents of gases and also among the most inert compounds that can be prepared. This type of product is described by J. G. Riess and M. Le Blanc, in xe2x80x9cBlood Substitutes: Preparation, Physiology and Medical Applications,xe2x80x9d Chap. 5 (K. C. Lowe, Ed., Ellis Horwood, Cichester, 1988), and by J. G. Riess in xe2x80x9cOrientations Actuelles en Matibre de Transporteurs d""Oxygene in vivo, les Emulsions de Fluorocarbures,xe2x80x9d J. Chim. Phys. 84(9), 1119-1127 (1987). Some fluorocarbon preparations can simultaneously fulfill yet other functions, e.g. as contrast agents in diagnosis or as drug carriers. However, the intravascular use of fluorocarbons requires that they be prepared in a dispersed form, for example, an emulsion, because fluorocarbons are not water-soluble. Such an emulsion usually comprises a fluorocarbon-based oxygen-carrier, one or more surfactants, water, and other ingredients, such as inorganic salts, to adjust the pH and the osmotic and oncotic pressures. Also the emulsion must comprise cryoprotectors if the emulsion is to be frozen. Other additives, such as nutritive agents, vitamins, steroids, prostaglandins, antibiotics, and thrombolytic agents may be required to meet specific therapeutic requirements.
In such emulsions, the fluorocarbons are dispersed in the form of droplets of about 0.2 xcexcm, by means of one or more surfactants. Different emulsions have been developed such as Fluosol-DA(copyright) or Fluosol(copyright), developed by the Green Cross Corporation in Japan, and the more concentrated fluorocarbon emulsions developed by Alliance Pharmaceutical, Inc. and described by C. Long, D. Long, J. G. Riess, R. Follana, A. Burgan and R. Mattrey in: xe2x80x9cBlood Substitutes,xe2x80x9d edited by T. M. S. Chang and R. P. Geyer (Marcel Dekker, Inc., New York, 1989), pp. 441-442. Certain disadvantages and/or limitations in emulsions of this type are described by J. G. Riess, C. Arlen, J. Greiner, M. Le Blanc, A. Manfredi, S. Pace, C. varescon and L. Zarif, in: xe2x80x9cBlood Substitutes,xe2x80x9d edited by T. M. S. Chang and R. P. Geyer (Marcel Dekker Inc., New York, 1989), pp. 421-430; and by J. G. Riess in Curr. Surg. 45, 365 (1988).
It is a fact that the surfactants used are polydisperse, and badly defined. Moreover, one of them, Pluronic F-68(copyright), the main surfactant in Fluosol-DA, causes a transitory anaphylactic reaction in certain patients. Further, the stability of Fluosol-type Pluronic F-68-based emulsions is limited; they must be frozen for storage and as manufactured, are not ready for use. Three preparations, the mother emulsion and two annex solutions, must be mixed before the emulsion is suitable for administration.
More generally, the properties and/or biocompatibility of the surfactants known and used so far are still insufficient to allow mastery of these emulsions, notably of their intravascular persistence and stability, and adaptation of their characteristics to a specific therapy. Still more generally, there is a scarcity of substances that are at the same time strongly amphtphilic and surfactive, biocompatible, and industrially feasible, while available at a reasonable cost.
Research has therefore been directed towards developing new surfactants or cosurfactants that are biocompatible and better adapted to the emulsification of fluorocarbons than those in use at present. The fluorinated derivatives of the invention improve such properties as described in U.S. Pat. No. 4,985,550 (Jul. 28, 1987) and U.S. Pat. No. 542,227 (Jun 6, 1990).
Other fluorinated compounds capable of improving the surfactive properties of fluorinated surfactants are described in U.S. Pat. No. 4,089,804. These compounds are formulated as (RF)nTmZ in which RF is a perfluoroalkyl chain, n=1 or 2, T is an alkylene, haloalkylene, arylene, alkylenethioalkylene, alkyleneoxyalkylene or alkyleneiminoaklylene chain, m=0, 1 or 2, and Z is a neutral or polar group such as COxe2x80x94NHxe2x80x94CH2OH.
These fluorinated compounds, in association with fluorinated surfactants, can find many applications, but their biocompatibility and toxicity is not assured. Moreover, they must always be used in conjunction with fluorinated surfactants.
Oligomers with perfluoroalkylated terminal groups are also described in EP-A-0 019 584, which can be used as surfactants and as additives in many products, in particular in extinctor foams. These too, then, are not intended for nor adapted to pharmaceutical use.
The invention provides amphiphilic fluorinated compounds of telomeric structure having the formula of I as described in the Detailed Description. The telomeric compounds comprise a fluorinated alkyl radical, RF the telogen, linked through an alkylenic group, X, to polymers of taxogens, or polymerizable unsaturated compounds, having the structures of III, a polyhydroxylated group, and V, an amino acid or peptide derivative, also described in the Detailed Description. RF may be joined as a telogen to a polymer of both III and V, or to a polymer of III alone.
In preferred embodiments, RF is the radical F(CF2)t-, wherein t=1 to 10; a preferred linear X is xe2x80x94CH2-CH2xe2x80x94, and a preferred branched X is 
The polyhydroxylated monomers XII from which the compounds of the invention are formed have the structure 
and in preferred embodiments, R1 is H or CH3, and R2, the polyhydroxylated group, is xe2x80x94C(CH2OH)3. In other preferred embodiments, R2 is a glucosyl, galactosyl, or glucaminyl moiety, or is xe2x80x94Zxe2x80x94R4xe2x80x94, wherein Z is a monovalent or bivalent radical selected from the group consisting of xe2x80x94NHxe2x80x94, xe2x80x94(CH2)rxe2x80x94Nxe2x80x94(R1)xe2x80x94, xe2x80x94(CH2)rxe2x80x94Oxe2x80x94, or (CH2)rxe2x80x94Sxe2x80x94, wherein r=2 to 4, and R4 is the lactobionyl, maltosyl or cellobiosyl radical.
The number of polyhydroxylated moieties in the surfactant is n, which can be from 1 to 50; in preferred embodiments, n is from 1 to 20.
The amino acid and peptide derivative monomers V from which the compounds of the invention are formed have the structure: 
wherein R1 is H or CH3, and R3 is a radical obtained from an amino acid or a peptide by removal of a hydrogen atom from the NH2 group thereof. The number of these amino acid derivative moieties in the surfactant is m, which can be 0 or from 1 to 200. These amino acid structure provide attachment sites for drugs and markers to the surfactant molecules, and in preferred embodiments, m is  greater than 1 and a portion of the m moieties are condensed with isothiocyanate to form a fluoresceinated molecule, as in tolomer 23 of Example 20. In a preferred embodiment of these fluorescein-labelled surfactant molecules, RF is C8F17 and X is xe2x80x94CH2xe2x80x94CH2xe2x80x94.
The invention also provides a method for synthesizing a telomeric compound of the invention wherein n is present and m=0, comprising reacting a monomer having the formula 
with a telogen of type RFXSH, under conditions serving to polymerize said monomer, comprising for example, the presence of a radical forming catalyst, initiator, or promotor, whereby the telomeric compound is formed.
The invention also provides a method for synthesizing a telomeric compound of the invention wherein n is present and m greater than 0, comprising reacting a monomer having the formula 
with a telogen of type RF-X-SH, under conditions serving to polymerize said monomer, comprising for example, the presence of a radical forming catalyst, initiator, or promotor, whereby the telomeric compound is formed.
According to another aspect of the invention there are provided formulations comprising any one of the amphiphilic fluorinated telomeric surfactants of the invention. Preferred formulations comprise at least one of these surfactants chemically bound to a biologically active substance or to a marker. The formulations can be in the form of a solution, gel, dispersion, including a liposomal formulation, an emulsion, or a microemulsion. In a particularly preferred embodiment, the amphiphilic fluorinated compound is incorporated into lipid vesicles or liposomes comprising natural or synthetic lipids. The formulations can be manufactured in a form suitable for the transport of gases, as a contrast agent, as a vehicle for the delivery of drugs, or for use as a marker. Formulations and emulsions can further comprise at least one highly fluorinated compound in addition to the amphiphilic fluorinated compound of the invention. In preferred embodiments, the highly fluorinated compound is selected from the group consisting of a bis(F-alkyl)-1,2-ethene; an F-isopropyl-1-F-hexyl-2-ethene; a bis (F-hexyl)-1,2-ethene; perfluorodecalin; perfluoromethydecalin; perfluorodimethydecalin; perfluoromethyladamantene or perfluorodimethyladamantene; a perfluorodi- or tri-methylbicyclo(3,3,1)nonane or a homologue thereof; an ether of the formula (cF3)2CFO(CF2CF2)2OCF(CF3)2; (CF3)2CFO(CF2CF2)3OCF(CF3)2; (CF3)2CFO(CF2CF2)2F; (CF3)2CFO(CF2CF2)3F; F(CF(CF3) CF2O)2CHFCF3; F(CF(CF3)CF2O)3CHFCF3, (C6F13)2O, (C4F9)2O, an amine selected from the group consisting of N(C3F7)3, a perfluoromethylquinolidine or perfluoromethyl-isoquinolidine, a halogenated derivative selected from the group consisting of C6F13Br, C6F13CBr2CH2Br, 1-bromo, 4-perfluoroisopropyl cyclohexane, and C8F16Br2. In particularly preferred embodiments, the highly fluorinated compound is selected from the group consisting of perfluorodecalin (F-decalin), bis (F-butyl)-1,2-ethene (F-44E) and heptadecafluorobromooctane, C8F,17Br (PFOB). In yet another preferred embodiment, the amphiphilic fluorinated compound is present in a concentration of from about 0.01 to 30% w/v. in an emulsion that further comprises a fluorinated or hydrocarbonated oil phase in a concentration of from about 10 to 125% w/v. The emulsion can further comprise at least a second amphiphilic compound. In preferred embodiments, the second amphiphilic compound is a lecithin or a polyoxyethylene polyoxypropylene copolymer.